Genetically stable oncolytic RNA virus, method of manufacturing and use thereof

ABSTRACT

In a method for manufacturing a modified enterovirus of ECHO 7 type by modification of native ECHO 7 virus, isolated by a known method from human feces and identified by genome sequence, the modification is performed initially conducting the virus adaptation in cancer cells, attenuated by anti-cancer agent dacarbazine, further passaging the modified virus in human embryonal fibroblast culture, followed by propagation in human melanoma cells and further passaging in human embryonal fibroblast culture, that was treated by ribavirin, isolation and purification by known method. The modified virus is suitable for treating various tumors.

TECHNICAL FIELD

The invention relates to development of a novel biotechnologicallyproduced anti-cancer preparation, namely to a genetically stableoncolytic RNA virus, a method for manufacturing the oncolytic virus, anduse thereof.

BACKGROUND ART

The ability of viruses to kill cancer cells is known for more than acentury [Kelly, E.; Russell, S. J. History of oncolytic viruses: genesisto genetic engineering. Mol. Ther. 2007, 15, pp. 651-659] and there werenumerous promising successes in experimental cancer therapy with variousviruses, nevertheless their use in clinical practice is hampered by thedifficulty to foresee the interaction between the tumour and its host,as well as the virus and response of human immune system to viralantibodies.

Although the clinical investigations regarding the use of viruses incancer therapy commenced more than 50 years ago, at present only twoviruses are approved for clinical use in cancer therapy. They areadenovirus with deleted E1B 55K gene (Garber, K. China approves world'sfirst oncolytic virus therapy for cancer treatment. J. Natl. CancerInst. 2006, 98., pp. 298-300) and unmodified passivized PicomaviridaeEnterovirus of Echo type (Eurasian patent 007839; European patentapplication 03733607), acting as antitumour immunostimulant.

Therefore, the development of novel efficient oncolytic viruses is stilla topical problem (Han Hsi Wong, Nicholas R. Lemoine,Yaohe Wang, Viruses2010, 2, pp. 78-106).

In order to increase the potential of virus so selectively infect cancercells and heighten the oncolytic activity, a number of modified viruseshave been disclosed. They are characterised by deletion of specificgenes, thus preventing their propagation in normal cells, or integrationof additional genes for improving the oncolytic properties.

However, the limited knowledge concerning the genetical modificationsthat provide for selectivity and efficiency against the tumour cells,results in modified viruses with lower cytolytic activity, compared toorigin, or higher anti-virus response of human immune system (S.Meerani,Yang Yao, Oncolytic viruses in cancer therapy. European Journal ofScientific Research, vol. 40 no. 1 (2010), pp. 156-171; Han Hsi Wong,Nicholas R. Lemoine,Yaohe Wang, Viruses 2010, 2, 78-106).

Although viruses are well-established tools for conveying vectors intocell, their use is limited by the high immunogenicity of viruses (Peng,Z. Current status of gendicine in China: recombinant human Ad-p53 agentfor treatment of cancers. Hum. Gene. Ther. 2005, 16, 1016-1027).

One of the most serious adverse properties of non-modified ECHO typeviruses, including ECHO 7, is their ability to cause infections that mayhave a fatal result (Wreghitt T. G., Gandy G. M., King A., Sutehall G.,Fatal neonatal ECHO 7 virus infection, The Lancet, vol. 324, p.465,1984). These viruses are known to be responsible for hand, foot andmouth disease in Malaysia (http://www.vadscomer.com/echovirus7.html),for myocarditis in leukemic child (Midula M., Marzetti G., Borra G.,Sabatino G., Myocarditis associated with ECHO 7 type infection inleukemic child, Acta Paediatrica Volume 65, Issue 4, pp. 649-651, July1976), aseptic meningitis, paralytic disease and fever(http://virology-online.com/viruses/Enteroviruses6.htm). Thereforepathogenicity is one of the major limitations that must be overcome inusing ECHO 7 type viruses in treating cancer patients.

DISCLOSURE OF THE INVENTION Technical Problem

Therefore, the problem to solve was the development a highly efficient,selective oncolytic virus without pathogenicity in normal cells and lowimmunological response, and possessing high genetic stability. It iswell known and recognised that RNA viruses mutate very easily uponpassage in cell cultures, which can change the phenotype, leading toincreased pathogenicity. Thus, for preparation of oncolytic virus-basedmedicine by using a wild non-pathogenic ECHO 7 virus strain as thestarting material, it is of extreme importance to find a procedure whichwould allow to generate an oncolytic modification of this virus thatwould retain non-pathogenic character of the original virus and begenetically stable.

Solution to the Problem

This problem was surprisingly solved by a targeted modification of asingle-strand RNA virus by developing a method that utilized the highmutation potential of single strand RNA virus in combination with aspecifically targeted selection of mutants, providing for fastseparation from the pool of mutant species with high and selectiveoncolytic activity. Many cancer cells are resistant to the virus (thevirus can not enter the cell and survive there). By careful selection ofcell lines where the virus is modified and by proper pre-treatment ofthe cancer cells it is possible to create a genetically stable andnon-pathogenic virus for cancer treatment. The virus provided by theinvention is in fact the first disclosure of a genetically stableoncolytic virus, based on ECHO-7 type virus, said genetically stablevirus bring usable for long term manufacturing (a multiple reproduction)as medicine.

SHORT DESCRIPTION OF THE INVENTION

We have developed a method for modifying the native ECHO 7 virus,identified by genome sequence SeqNo2, the method comprising initiallyconducting the virus adaptation in cancer cells, attenuated by ananti-cancer agent such as dacarbazine, passaging the modified virus inhuman embryonal fibroblast culture, propagation in human melanoma cellsand passaging in human embryonal fibroblast culture, optionally treatedby ribavirin, isolation of the virus and purification of the virus. Thevirus can be isolated and purified by known methods. The use ofanticancer agent such as dacarbazine in subtoxic concentrations formodification of cancer cells and using these treated cancer cells ashost cells for virus replication has led to creation of mutant viruswith stable genome applicable as highly effective medicine for treatmentof cancer.

More than one type of cell lines can be used during conducting the virusadaptation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a comparison of genomes of the modified virus (Seq ID No 1)and unmodified (native) virus (Seq ID No 2), and

FIG. 2 is a comparison of amino acid sequences of the modified virus(Seq ID No 4) and unmodified (native) virus (Seq ID No 5).

SEQUENCE LISTING FREE TEXT

Seq ID No 1: Modified virus;

Seq ID No 2: Unmodified (native) virus;

Seq ID No 3: Modified virus after propagation for 12 months;

Seq ID No 4: Amino acid sequence of the modified virus;

Seq ID No 5: Amino acid sequence of the unmodified virus;

Seq ID No 6: Primer Eo7-1F; Seq ID No 7: Primer Eo7-1R;

Seq ID No 8: Primer Eo7-2F; Seq ID No 9: Primer Eo7-2R;

Seq ID No 10: Primer Eo7-3F; Seq ID No 11: Primer Eo7-3R;

Seq ID No 12: Primer Eo7-4F; Seq ID No 13: Primer Eo7-4R;

Seq ID No 14: Primer Eo7-5F; Seq ID No 15: Primer Eo7-5R;

Seq ID No 16: Primer Eo7-6F; Seq ID No 17: Primer Eo7-6R;

Seq ID No 18: Primer Eo7-7F; Seq ID No 19: Primer Eo7-7R;

Seq ID No 20: Primer Eo7-8F; Seq ID No 21: Primer Eo7-8R;

Seq ID No 22: Primer Eo7-9F; Seq ID No 23: Primer Eo7-10F;

Seq ID No 24: Primer Eo7-9R; Seq ID No 25: Primer Eo7-11F;

Seq ID No 26: Primer Eo7-11R; Seq ID No 27: Primer Eo7-12F;

Seq ID No 28: Primer Eo7-12R; Seq ID No 29: Primer Eo7-13F;

Seq ID No 30: Primer Eo7-13R; Seq ID No 31: Primer Eo7-14F;

Seq ID No 32: Primer Eo7-14R; Seq ID No 33: Primer Eo7-15F;

Seq ID No 34: Primer Eo7-15R; Seq ID No 35: Primer Eo7-16F;

Seq ID No 36: Primer Eo7-17F; Seq ID No 37: Primer Eo7-16R;

Seq ID No 38: Primer Eo7-18F; Seq ID No 39: Primer Eo7-18R;

Seq ID No 40: Primer Eo7-19F; Seq ID No 41: Primer Eo7-19R;

Seq ID No 42: Primer Eo7-20F; Seq ID No 43: Primer Eo7-20R;

Seq ID No 44: Primer Eo7-21F; Seq ID No 45: Primer Eo7-21R;

Seq ID No 46: Primer Eo7-22F; Seq ID No 47: Primer Eo7-22R;

Seq ID No 48: Primer Eo7-23F; Seq ID No 49: Primer Eo7-23R;

Seq ID No 50: Primer Eo7-24F; Seq ID No 51: Primer Eo7-24R;

Seq ID No 52: Primer Eo7-25F; Seq ID No 53: Primer Eo7-25R;

Seq ID No 54: Primer Eo7-26F; Seq ID No 55: Primer Eo7-26R.

DETAILED DESCRIPTION OF THE INVENTION

We have unexpectedly discovered the suitability for this purpose of aknown Echo 7 type Picomrnaviridae enterovirus, isolated from a humanintestine. The original nucleotide sequence, determined by a standardmethod, was found to be rather similar to that of Wallace typePicomaviridae Enterovirus.

Checking the oncolytic activity of isolated native enteroviruses intissue of angiosarcoma demonstrated that neither individual viruses northeir combinations in a dose 3×10⁵ TCID₅₀/0.03 ml possessed substantialoncolytic activity with exception of ECHO 7 type that showed morepromising activity (Table 1).

TABLE 1 Influence of viruses on angiosarcoma tissue culture Number ofregressed Viral titer on Number tumours Isolated Day 4, of on Day 4(surviving) TCD₅₀/ Virus animals after infecting virus 0.1 ml ECHO 4 6 0ECHO 4 10⁶-10⁷ ECHO 7 6 0 ECHO 7 10⁵-10⁶ Coxsackie B-5 6 0 Coxsackie B-510⁷-10⁸ ECHO 4 + 6 2 ECHO 7 10⁹ ECHO 7 ECHO 4 + 6 1 ECHO 4, 10³Coxsackie B-5 Coxsackie B-5 {close oversize brace} 10⁸ ECHO 7 + 6 0 ECHO7 10⁴ Coxsackie B-5 Control 6 0 10⁸

The instability of the genome of the RNA single strand viruses is awell-known fact; therefore, such viruses usually are not selected forconstructing oncolytic viral agents.

The modification of the isolated native virus was realised in severalconsecutive steps.

The first step takes advantage of the high mutation potential of RNAviruses (on average one mutation on each replication) to develop acytopathic mutant by replicating the virus in trypsinized monolayer ofhuman embryonic fibroblast culture in presence of calf serum.

Cells were incubated for 10 days, carrying out the passage each timewhen the cells in culture had degenerated for 50%.

Testing the selected virus in RD cell culture a pronounced cytopathiceffect was observed already in 24 hours after infection. The titer ofthe developed virus, determined by last dilution method wasTCID₆₀=1×10⁻⁸.

This strain was propagated, isolated and stored at ˜70° C. for furtheruse in producing selective and genetically stable oncolytic strain.

The virus so obtained was modified, using specially developed methodcomprising three steps.

1st Modification Step in a First Tumour Cell Line

In the first modification step, the virus was propagated in tumour celllines attenuated by anticancer agent. Human breast adenocarcinoma cellline (MCF-7) was used in this step.

A monolayer of these cells was treated with dacarbazine DTIC in subtoxic dose (20 μM). After treating with dacarbazine, the cells weretransferred to fresh culture medium and contacted with the virus, andthe propagation continued without adding serum. After 24 hours fromcontacting with the virus, the cells were removed and the virus wasisolated from the media.

The virus was repeatedly propagated (passaged) in human embryonicfibroblast cell culture and again used for infecting the MCF-7 cellline. This procedure was repeated 10 times. Thus, this modification stepcomprises alternately propagating the virus in human breastadenocarcinoma cells and human embryonic fibroblast cells.

2nd Modification Step in a Second Tumour Cell Line

In the next modification step, the virus as described above, wascontacted with gastric adenocarcinoma cell culture. A monolayer of thesecells was treated with dacarbazine DTIC in sub toxic dose (20 μM).

The monolayer of these cells was infected by the virus, which wasisolated after the modification in the first step, and the propagationcontinued in a culture medium without serum.

After 24 hours from contacting with the virus, the cells were removedand virus isolated from the media. The virus was repeatedly propagated(passaged) in human embryonic fibroblast cell culture, and thereafteragain used for infecting the gastric adenocarcinoma cell line. Thisprocedure was repeated 10 times. Thus, this second modification stepcomprises alternately propagating the virus in gastric adenocarcinomacells and in human embryonic fibroblast cells.

In the first modification step and in the second modification step, thepropagation procedure was always finished by propagating the virus lastin the human embryonic fibroblast culture.

3rd Modification Step in Human Melanoma Cells

The virus produced in the second step was used for infecting humantumours, obtained in surgery.

Melanoma cancer tissues were obtained in surgery from 23 patientspreviously treated by chemotherapy.

Tissues were infected by the modified virus and incubated at 37° C. inthe absence of carbon dioxide. Before being used for infecting a newtissue material (fresh melanoma tissue from another patient), themodified virus was repeatedly propagated in human embryonic fibroblastculture to titer 7 Ig TCID₅₀/1 ml.

The modified virus was propagated in human embryonic fibroblast cellculture that was treated by 5 mM ribavirin 7 hours before infection andcultivated for 24 hours. The virus was isolated from the culture, andthe procedure of propagating the virus in the fibroblast culture wasrepeated 10 times.

Finally, the virus was isolated, purified and propagated in humanembryonic fibroblast culture without addition of ribavirin.

S The propagated virus was used for sequence determination. The genomeof the modified virus differs from that of frozen unmodified nativevirus (Echo 7 type Wallace strain from NCBI database) for about 10%, thecoat part for about 12%.

The virus modified by the described method was found to be surprisinglystable. Its genome changed for only 0.7% after continuous passaging for12 months (propagation for 12 months in human embryonal lung culture MRC5).

Especially important is the fact that the modified virus (further on MV)is characterised by exceptionally high cytopathic effect on malignantcells and low cytotoxicity on normal cell lines as well as no toxicityin vivo in mice.

In experiments with cell lines MV was found to be cytotoxic for melanomacell lines FM9, FM55, FM94 and SK-Me126, gastric carcinoma cells, humanoral squamous cell carcinoma SCC25 cells, human epithelial cell linederived from a lung carcinoma (A549), acute monocyte leukemia THP-1cells, rabdomyosarcoma RD cells, human pancreatic adenocarcinoma HPAF-IIcells, human breast adenocarcinoma cells (MCF-7) as well as on primarycell cultures of gastric adenocarcinoma GC1 and thyroid cancer lineHA007.

In animal experiments, MV caused regression of murine sarcoma M-1, micefibrosarcoma MX-17 as well as transplantable tumours—Moloney sarcoma(SM) and KRS-321 sarcoma.

In a clinical pilot study, a group of 46 melanoma stage I patients noprogress of melanoma was observed for 50 months in 43 patients, treatedwith MV. In the control group, melanoma progressed for 10 of 31 patientsundergoing standard therapy.

In a 50 months study of 44 stage II melanoma patients the progress ofmelanoma was stopped in 38 patients, compared to control group of 36patients undergoing standard therapy, where melanoma did not progress in15 patients, but did progress in 21 patient. No serious adverse effectswere observed for patients treated with MV.

INDUSTRIAL APPLICABILITY

We have developed a novel virus strain (MV) with original genomesequence, stable against genetic drift, possessing cytopathic activityagainst various types of tumours, characterized by low incidence ofadverse effects and low toxicity that can be used with advantage incancer virotherapy. Thus, we have unexpectedly solved the main obstaclein wider use of RNA viruses in medicine—obtained genetically stablestrain that can be used in standardized continuous manufacturing ofoncolytic viral preparation. The viral preparation can be used inanticancer therapy against a variety of tumour cells.

EXAMPLES

The present invention is described in Examples in more detail. However,the invention is not construed as being limited to the examples.

Virus

The virus modified according to the invention is ECHO-7 virus(Picomaviridae family, Enterovirus genus, ECHO (Enteric Cytopathic HumanOrphan) type 7, group IV, positive-sense single stranded RNA virus). Thenative virus can be identified by genome sequence Seq Id No 2.

Example 1. The Isolation and Characterization of the Original VirusStrain

A known method for isolation (A. C. Rentz, J. E. Libbey, R. S. Fujinami,F. G. Whitby, and C. L. Byington. Investigation of Treatment Failure inNeonatal Echovirus 7 Infection. The Pediatric Infectious DiseaseJournal, Volume 25, Number 3, March 2006, 259) and propagation in BS-C-1cell line (CCL 26; ATCC) was used (Libbey J E, McCright I J, Tsunoda I,et al. Peripheral nerve protein, P0, as a potential receptor forTheiler's murine encephalomyelitis virus. J Neurovirol. 2001; 7:97-104.Pevear D C, Tull T M, Seipel M E, et al. Activity of pleconaril againstenteroviruses. Antimicrob Agents Chemother. 1999; 43:2109-2115). Viruspropagation and determination of titer was conducted in concordance withthe published method (Zurbriggen A, Fujinami R S. Aneutralization-resistant Theiler's virus variant produces an altereddisease pattern in the mouse central nervous system. J Virol. 1989;63:1505-1513).

Example 2. Virus Modification

In the first modification step, the virus was propagated in tumour celllines attenuated by an anticancer agent. Initially, for propagation wasused the human breast adenocarcinoma cell culture (MCF-7), cultivated inDME medium (Sigma-Aldrich) with 10% serum (Gibco) and antibiotics (100IU/mi penicillin, 100 IU/ml streptomycin) at 37° C. under atmosphere,containing 5% CO₂ until developing of the monolayer.

The obtained monolayer of these cells was treated with dacarbazine DTICin sub toxic dose (20 μM). After treating with dacarbazine cells weretransferred to fresh culture medium without added serum, the cellscontacted with virus and the propagation continued.

After 24 hours from contacting with the virus the cells were removed andvirus isolated from the media. The virus was repeatedly propagated inhuman embryonal fibroblast cell culture and again used for infecting theMCF-7 cell line. This procedure was repeated 10 times.

In the next, second step, the virus as described above, was contactedwith gastric adenocarcinoma cell culture. The cell culture forpropagation was cultivated in DME medium (Sigma-Aldrich) with 10% serum(Gibco) and antibiotics (100 IU/ml penicillin, 100 IU/mi streptomycin)at 37° C. under atmosphere, containing 5% CO₂ until developing of themonolayer.

The obtained monolayer of these cells was treated with dacarbazine DTICin sub toxic dose (20 μM). After treating with dacarbazine cells weretransferred to fresh culture medium without added serum, the cellscontacted with virus and the propagation continued.

After 24 hours from contacting with the virus the cells were removed andvirus isolated from the media. The virus was repeatedly propagated inhuman embryonal fibroblast cell culture and again used for infecting thegastric adenocarcinoma cell line. This procedure was repeated 10 times.

In the third step, the virus produced in the second step was used forinfecting human tumours, obtained in surgery. Melanoma cancer tissueswere obtained in surgery from 23 patients previously treated bychemotherapy.

The tumour cells were separated from fat cells, necrotic tissue andblood, kept at 0° C. for 24 hours, fragmented and as approximately 0.1cm³ large tissue pieces immersed in Eagle medium (4 mi of medium for 10mg of tissue), infected with the prepared virus and incubated in theabsence of carbon dioxide at 37° C.

The medium was replaced by a fresh portion every day until thedestruction of tumour, determined morphologically and visually by theoxidation level of medium.

The virus titer was determined every day in tumor tissue fee mediumsample. The reproduction rate of virus was determined from the virustiter at the conclusion of an experiment in comparison with that on Day0. Such modification of virus was performed in tissues obtained from 23patients.

Before being used for infecting a new tissue material, the modifiedvirus was each time repeatedly propagated in human embryonal fibroblastculture to titer 7 Ig TCID₅₀/1 ml.

The modified virus was propagated in human embryonal fibroblast cellculture that was treated by 5 mM ribavirin 7 hours before infection andcultivated for 24 hours. Virus was isolated from culture medium, and theprocedure repeated 10 times.

Finally, the virus was isolated, purified and propagated in humanembryonal fibroblast culture without addition of ribavirin.

The propagated virus sample was used for determination of genomesequence, anticancer activity and replicative stability by passaging itfor 12 months in human embryonal fibroblast culture with repeateddetermination of genome sequence (Seq ID No 1).

Example 3. Determination of Virus Genome Sequence

The isolation, amplification and sequencing of the isolated, modifiedand cultivated virus genome were performed according to the known method[Chua B H, McMinn P C, Lam S K, Chua K B. Comparison of the completenucleotide sequences of echovirus 7 strains UMMC and the prototype(Wallace) strain demonstrates significant genetic drift over time. J GenVirol. 2001 November; 82(Pt 11): 2629-39].

For this purpose, 96 enteroviruses with complete genome sequence wereselected from the NCBI Gene bank. The complete genome sequences forthese viruses were downloaded and compared by Vector NTI program. Basedon the results of comparing the most conservative regions of virusgenomes were determined and 13 degenerated oligonucleotide pairsselected in these regions, covering the length of the potentialenteroviruses genome. After the synthesis of the first 13 fragments,another 13 nucleotide pairs were produced. These oligonucleotide pairswere virus-specific and designed so as to produce overlaying fragments.After the building of the full genome sequence the virus genome wasrepeatedly sequenced with the virus-specific primers.

Example 3.1. The Genome Sequence of the Unmodified (Native) Virus

The sequence of the native virus was produced from 26 separateoverlapping PCR fragments, synthesized from the primers listed in Table2.

TABLE 2 Primers used to sequence the complete genome of viruses. lengthTarget Primer Sequence (5′-3′) (bp) Position region Eo7-1FTTAAAACAGCCTGTGGGTTG 20    1-20 5′UTR Eo7-1R GAAACACGGACACCCAAAGTAG 22 545-566 5′UTR Eo7-2F CCATGGGACGCTTCAATACT 20  391-410 5′UTR Eo7-2RGCACCAGTCTTTTGTGTCGA 20  758-777 VP4 Eo7-3F CGACTACTTTGGGTGTCCGTGTTTC 25 542-566 5′UTR Eo7-3R TCDGGRAAYTTCCACCACCACCC 23 1178-1200 VP2 Eo7-4FCGACAGGGTGAGTCCCTAA 20  979-998 VP2 Eo7-4R TTTCACCCTTCGTGAGGTTC 201381-1400 VP2 Eo7-5F GCATCYAARTTYCAYCARGG 20 1289-1308 VP2 Eo7-5RCACATKGGKGCAATSGTGAC 20 1676-1695 VP2 Eo7-6F GTGGATCAACTTGCGCACTA 201513-1532 VP2 Eo7-6R AAATTGTGGCATAGCCGAAG 20 1797-1816 VP3 Eo7-7FGTCACSATTGCMCCMATGTG 20 1676-1695 VP2 Eo7-7R CTTNATRCTYCCTGACCAGTGTG 232055-2077 VP3 Eo7-8F AAGCATGGACGCATATCACA 20 1921-1940 VP3 Eo7-8RGATATGGGTTCCCACATTGC 20 2174-2194 VP3 Eo7-9F CACACTGGTCAGGRAGYATNAAG 232055-2077 VP3 Eo7-10F CAAGTGTGTCGTCCTGTGCT 20 2350-2369 VP3 Eo7-9RCCTATTGGCGCTGTCTTGAT 20 2694-2713 VP1 Eo7-11F ACCAAAGATCAAGACAGCGC 202687-2706 VP1 Eo7-11R TTGGCACCCACACTCTGATA 20 3178-3197 VP1 Eo7-12FACCAGTCCGGTGCTGTTTAC 20 3336-3355 VP1-2A Eo7-12R TCCCAYACACARTTYTGCCAGTC23 3401-3423 2A Eo7-13F CARAAYTGTGTGTGGGAAGACTA 23 3407-3429 2A Eo7-13RCCCTGYTCCATKGCTTCATCYTCYARC 27 3748-3774 2A-2B Eo7-14FTTACCCAGTCACCTTCGAGG 20 3535-3554 2A Eo7-14R TGTTTTTCCTTCACTTCCGG 204181-4200 2C Eo7-15F GTTRGARGATGATGCNATGGARCARGG 27 3748-3774 2A-2BEo7-15R TCAATACGGYRTTTGSWCTTGAA 23 4409-4431 2C Eo7-16FCCTYTRTAYGCVGCYGARGC 20 4343-4362 2C Eo7-17F TTCAGWSCAAAYRCCGTATTGA 234409-4431 2C Eo7-16R AAYTGAATGGCCTTHCCACACAC 23 4922-4944 2C Eo7-18FCTDGTGTGTGGRAAGGCYATNCA 23 4919-4941 2C Eo7-18R TATGCTCCYTGRAARCCTGCAAA23 5309-5330 3A-3B Eo7-19F CAAGCCCTAACCACGTTTGT 20 5252-5271 3A Eo7-19RACCCGTAGTCAGTCACCTGG 20 5740-5759 3C Eo7-20F TTTGCAGGMTTYCARGGWGCATA 235309-5330 3A-3B Eo7-20R GCYCTWGTGGGRAAGTTRTACAT 23 5723-5745 3C Eo7-21FGTGTTGGATGCCAAGGAACT 20 5555-5574 3C Eo7-21R ATGGGCTCCGATCTGATGTC 206203-6222 3D Eo7-22F TTCCCCACWAGRGCAGGCCARTGYGG 26 5907-5832 3C Eo7-22RCTCCAAAAGASRTCYGGGTCRCA 23 6572-6594 3D Eo7-23F TGAAGGATGCATGGACAAA 206360-6379 3D Eo7-23R ATGGGTATTGCTCATCTGCC 20 7078-7097 3D Eo7-24FTGYGACCCRGAYSTVTTTTGGAG 23 6572-6594 3D Eo7-24R TCRTGDATDTCYTTCATGGGCA22 7116-7137 3D Eo7-25F CCTGGACGAATGTGACCTTT 20 7041-7060 3D Eo7-25RCCCTACCGCACTTTTATCCA 20 7384-7403 3′UTR Eo7-26F ATCCAYGARTCHATYAGRTGGAC23 7130-7152 3D Eo7-26R CCGCACCGAATGCGGAGAATTTAC 24 7404-7427 3′UTRUTR-untranslated region.

The 5′-terminal and the 3′-terminal sequences were obtained, using5′-RACE and 3′-RACE methods, correspondingly.

As a result, the full genome sequence of the unmodified virus was foundto consist of 7434 nucleotides, excluding the poly A sequence (Seq ID No2). The untranslatable 5′-terminal (5′NTR) contains 742 nucleotides,followed by coding part starting with start codon (AUG) at position 743,containing codons for 2196 amino acids and ending with stop codon (UAA)at position 7331 (Seq ID No 2). The untranslatable 3′-terminal (3′NTR)of this strain contains 100 nucleotides, followed by poly A sequence.

Example 3.2. The Sequence of the Modified Virus (MV)

The sequence of the starting virus was produced from 26 separateoverlapping PCR fragments, synthesized using the primers listed in Table2.

The 5′-terminal and the 3′-terminal sequences were obtained, using5′-RACE and 3′-RACE methods, correspondingly.

As a result, the full genome sequence of the modified virus was found toconsist of 7427 nucleotides, excluding the poly A sequence (Seq ID No1).

The untranslatable 5′-terminal (5′NTR) of this strain contains 742nucleotides, followed by the coding sequence. The coding part thatcontains information about the virus polyprotein, begins with the startcodon (AUG) at position 743, contains codons for 2194 amino acids andends with stop codon (UAA) at position 7325 (Seq ID No 1). Theuntranslatable 3′-terminal (3′NTR) of this strain contains 100nucleotides, followed by poly A sequence.

Example 3.3. The Genome Sequence of the Modified Virus after Propagationfor 12 Months

The sequence of the modified virus was produced from 26 separateoverlapping PCR fragments, synthesized the primers listed in Table 2.

The 5′-terminal and the 3′-terminal sequences of this strain wereobtained, using 5′-RACE and 3′-RACE methods, correspondingly.

As a result, the full genome sequence of the modified virus was found toconsist of 7427 nucleotides, excluding the poly a sequence (Seq ID No3). The untranslatable 5′-terminal (5′NTR) contains 742 nucleotides,followed by coding part, starting with start codon (AUG) at position743, containing codons for 2194 amino acids and ending with stop codon(UAA) at position 7325 (Seq ID No 3). The untranslatable 3′-terminal(3′NTR) of this strain contains 100 nucleotides, followed by poly Asequence.

Example 3.4. Comparison of Genomes of Modified Virus (MV) and NativeStrain

Comparison of genomes of modified virus (MV) and starting strain isprovided in FIG. 1.

The difference in nucleotide sequence, calculated by programme VectorNTI is substantial, 10% for the complete genome and 12% for the partcoding the virus coat proteins. The amino acid sequences for themodified and starting strains are listed in FIG. 2.

Example 3.5. The Genome Sequence of the Modified Virus after Propagationfor 12 Months

S The changes in the sequence of modified virus (MV) genome aftercontinuous passaging for 12 months did not exceed 0.7% of the initialsequence. All found changes were one nucleotide replacements, partiallythe mute mutations (without change of amino acid). If the amino acid waschanged, its position was in the genome polymorphic part, evidentlywithout relevant influence on virus activity.

Example 4. Virus Passaging

Virus MV was passaged by known methods and propagated for 12 months inhuman embryonal lung culture MRC 5 (Instituto ZooprofilatticoSperimentale della Lombardia e dell Emilia, Brescia—Laboratorio CentroSubstrati Cellulari, Catalogue No. BS CL 68 (origin: American TypeCulture centre Collection, Rockville, Md., USA), free of bacteria,viruses, fungi or mycoplasmas, and later stored frozen at −70° C.

Example 5. Determination of Anti-cancer Activity of the Modified Virus(MV)

In experiments with cell lines, MV was found to cytotoxic for melanomacell lines FM9, FM55, FM94 and SK-Me126, gastric carcinoma cells, humanoral squamous cell carcinoma SCC25 cells, human epithelial cell linederived from a lung carcinoma (A549), acute monocyte leukemia THP-1cells, rabdomiosarcoma RD cells, human pancreatic adenocarcinoma HPAF-IIcells, human breast adenocarcinoma cells (MCF-7) as well as on primarycell cultures of gastric adenocarcinoma GC1 and thyroid cancer lineHA007. Thus, for example, MV injections for 3 days caused reducing ofsarcoma M-1 mass in 55% (in 11 of 22) of animals, compared with 6% (in 1of 18) spontaneous regression in the control group.

Transplanting sarcoma KRS-321 on Day 5 after the injecting MV in a dose15×10⁶ TCID₅₀ on Wistar rats in 44% of animals (11/25) the regression oftumour was observed, while in the control group there were no cases ofregression.

Testing the anti-cancer activity of the virus sample after the 12 monthspassaging on the same cancer cell lines and transplanted tumours nostatistically significant difference from the original MV was observed.Neither MV nor the virus passaged for 12 months caused any toxicreactions in intact mice.

Example 6. Anti-cancer Activity of Modified Virus in Treating Patients

Treating of melanoma patients by the modified virus (MV) was conductedaccording to the following scheme: therapy was commenced 2-3 weeks afterthe excision of the tumour by intramuscular administration of 2 ml ofsolution with titer 2×10⁶ TCID₅₀/ml—2×10⁸ TCID₅₀/ml for 3 daysconsecutively with supporting injections at monthly intervals accordingto the same 3 day schedule. After the fourth month, the viruspreparation was administered once monthly for the next 8 months. In thenext 2 years the supporting therapy was continued with the same dose,gradually increasing the interval between administrations to 6, 8 and 12weeks.

In a clinical pilot study, a group of 46 melanoma stage I patients noprogress of melanoma was observed for 50 months in 43 patients, treatedwith MV. In the control group, melanoma progressed for 10 of 31 patientsundergoing standard therapy.

In a 50 months study of 44 stage II melanoma patients the progress ofmelanoma was stopped in 38 patients, compared to control group of 36patients undergoing standard therapy, where melanoma did not progress in15 patients, but did progress in 21 patients.

The efficiency of treatment is characterized by the following examples:

Case 1. Female, age 76, Melanoma cutis dorsi

Op. 11 Sep. 2009. Excisio tu cutis dorsi

pT4b N0 M0

SN biopsy was not performed

Ex consilio: follow-up

Op. 7 Apr. 2010. LAE axilaris sin.

Mts l/n axilarns sin

Ex consiio: Roferon

Roferon 6 mil 3× per week from 24 Jun. 2010 till 30 Aug. 2010.

The treatment was discontinued due to the side effects.

From October 2010 the therapy with virus preparation in 2 ml dose withtiter 2×10⁶ TCID₅₀/ml—2×10⁸ TCID₅₀/ml was commenced. The treatment waswell tolerated, and no progression of the disease was documented untilJan. 2, 2012.

Case 2. Female, age 42, Melanoma cutis dorsi

Op. 25 May 2008. Excisio tu cutis dorsi

pT4a N0 M0, Clark V, Breslow 9 mm

SN biopsy was not performed

Virus preparation (2 ml with titer 2×10⁶ TCID₅₀/ml—2×10⁶ TCID₅₀/ml wasadministered from 27 Jun. 2008 till 27 Jun. 2011.

21 Jan. 2011. US examination: recurrence in the scar

Op. Feb. 2, 2011. Excisio. Histological examination: granuloma.

Virus preparation (2 ml with titer 2×10⁶ TCID₅₀/ml—2×10⁸ TCID₅₀/ml was20 continued till 27 Jun. 2011.

During the observation period (till December 2011) no evidence of thedisease progression was documented.

Case 3. Female, age 57, Melanoma cutis dorsi

Op.19 Aug. 2007. Excisio tu cutis dorsi

P T3b N0 M0

SN biopsy was not performed

Recommendations: follow-up

Op. 10 Dec. 2009. LAE colli dx. Histological examination: mts I/n collidx

Progression of the disease—US examination on 22 Feb. 2010: mts Vn colli

22 Feb. 2010. Ex consilio: no surgery was recommended due to bulkydisease

Virus preparation (2 ml with titer 2×10⁶ TCID₅₀/ml—2×10⁸ TCID₅₀/ml was

administered from 22 Feb. 2010 and still is in progress.

Last visit at clinic on 22 Nov. 2011—the disease has stabilized.

Case 4. Female, age 58, Melanoma cutis dorsi

Op. April 2004. Excisio tu cutis dorsi, LAE axillaris sin.

pT4b, N2c, M0 (Breslow 15 mm)

Reexcisio January 2006, September 2006 (local recurrence)

Therapy with IFN from October 2006 till May 2007.

Reexcisio cum dermoplasticum February 2007, May 2007, September 2007.

Virus preparation (2 ml with titer 2×10⁶ TCID₅₀/ml—2×10⁸ TCID₅₀/ml wasadministrated from February 2008 till April 2011.

Visceral metastasis February 2011.

Exitus letalis October 2011.

Dose Form and Administration

The viral preparation for therapeutic treatment can be in the form ofinjectable aqueous solution containing the modified virus having thestable genome sequence as explained above, for example in the titer of2×10⁶ TCID₅₀/ml—2×10⁸ TCID₅₀/ml. The solution carrying the virus can beany physiologically acceptable sterile solution, especially sodiumchloride solution. The preparation is stored and transported in frozencondition and defrozen at room temperature before the use. Thepreparation can be in vials or other container units in volumes thatcorrespond a single dose injected at a time to the patient.

The preparation can be administered by injecting it intramuscularly(i.m.) to the patient after the excision of the tumour in question, whenthe wound has healed. The dosage can be 2 ml of the above-mentionedsolution at a time The intramuscular administration by injection isrepeated according to the planned therapy schedule.

What is claimed:
 1. A method of treating a patient suffering from anoncological disease by administering into said patient apharmaceutically effective amount of a modified enterovirus of the ECHO7 type having the genome sequence of SEQ ID No. 1 or a sequence havingat least 99% sequence identity with SEQ ID No.
 1. 2. The method of claim1, wherein the oncological disease is selected from the group consistingof: melanoma, gastric cancer, intestinal cancer, human breast cancer,prostate cancer, pancreatic cancer, lung cancer, kidney cancer, bladdercancer, lymphosarcoma, uterine cancer, angiosarcoma, rhabdomyosarcoma.3. The method of claim 1, wherein the oncological disease is melanoma.4. The method of claim 1, wherein the patient is a human.
 5. The methodof claim 1, wherein the enterovirus is administered by intramuscularadministration.
 6. The method of claim 1, wherein the enterovirus isadministered with a titer of 2×10⁶ to 4×10⁸ TCID₅₀/mL.